ãŽâ² actin  (Millipore)


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  • 99
    Name:
    Bismarck Brown R
    Description:
    Bismark Brown R BB also called as basic brown 4 belongs to the class of diazo dyes It has the absorption maximum at 468nm
    Catalog Number:
    15000
    Price:
    None
    Applications:
    Bismarck Brown R has been used as an adsorbate and is used in dye decoloration experiment.
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    Structured Review

    Millipore ãŽâ² actin
    Bismarck Brown R
    Bismark Brown R BB also called as basic brown 4 belongs to the class of diazo dyes It has the absorption maximum at 468nm
    https://www.bioz.com/result/ãŽâ² actin/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ãŽâ² actin - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma"

    Article Title: Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-18-0086

    TGFβ inhibition augments anti–CTLA-4 immunotherapy in an autochthonous BRAF V600E PTEN -/- melanoma model. A. Mice were treated with either anti–CTLA-4 (purple, 100 μg i.p.) or IgG isotype control (black, 100 μg i.p.) every 3 days when tumors reached 60–80 mm 3 . Left : tumor volumes monitored every 3 days. Right : representative tumor photos. 6 mice/group. Representative of 3 independent experiments. B. Left : resected Braf V600E Pten -/- melanoma tissues analyzed for TGFβ1 expression by IHC and whole tissue Western blots. IHC representative of 3 tumor specimens (40x). Right : Spearman correlation between tumor volume and TGFβ1/β-actin density ratios from Western blots. C. Phospho-SMAD2 (pSMAD2) Western blot analysis performed following TEW-7197 treatment (25 mg/kg daily p.o. x 2 weeks). tSMAD2: total SMAD2. Representative of 2 independent experiments. D. Left : mice were treated with TEW-7197 monotherapy (25 mg/kg p.o. daily). Tumor volumes were monitored every 3 days. 6 mice/group. Representative of 3 independent experiments. Center : final tumor weights. Right : representative photos of resected tumors. ns: non-significant. E. Left : mice were treated with either IgG isotype control/vehicle control, TEW-7197(25 mg/kg p.o. daily) monotherapy, or combination anti–CTLA-4 (100 μg i.p. every 3 days)/TEW-7197. Left : tumor volumes monitored every 3 days. 6 mice/group. Black arrow, treatment initiation. Representative of 2 independent experiments. Right top : lungs resected and enumerated for metastatic foci by staggered IHC. Right bottom : Kaplan-Meier survival plot. Data analyzed by the log-rank test. F. Left : flow cytometry on tumor-infiltrating lymphocytes (TILs) at the conclusion of the experiment. 5 tumors/group. Right . All data is mean±SEM. Significance calculated using the unpaired t-test or a one-way ANOVA. * p
    Figure Legend Snippet: TGFβ inhibition augments anti–CTLA-4 immunotherapy in an autochthonous BRAF V600E PTEN -/- melanoma model. A. Mice were treated with either anti–CTLA-4 (purple, 100 μg i.p.) or IgG isotype control (black, 100 μg i.p.) every 3 days when tumors reached 60–80 mm 3 . Left : tumor volumes monitored every 3 days. Right : representative tumor photos. 6 mice/group. Representative of 3 independent experiments. B. Left : resected Braf V600E Pten -/- melanoma tissues analyzed for TGFβ1 expression by IHC and whole tissue Western blots. IHC representative of 3 tumor specimens (40x). Right : Spearman correlation between tumor volume and TGFβ1/β-actin density ratios from Western blots. C. Phospho-SMAD2 (pSMAD2) Western blot analysis performed following TEW-7197 treatment (25 mg/kg daily p.o. x 2 weeks). tSMAD2: total SMAD2. Representative of 2 independent experiments. D. Left : mice were treated with TEW-7197 monotherapy (25 mg/kg p.o. daily). Tumor volumes were monitored every 3 days. 6 mice/group. Representative of 3 independent experiments. Center : final tumor weights. Right : representative photos of resected tumors. ns: non-significant. E. Left : mice were treated with either IgG isotype control/vehicle control, TEW-7197(25 mg/kg p.o. daily) monotherapy, or combination anti–CTLA-4 (100 μg i.p. every 3 days)/TEW-7197. Left : tumor volumes monitored every 3 days. 6 mice/group. Black arrow, treatment initiation. Representative of 2 independent experiments. Right top : lungs resected and enumerated for metastatic foci by staggered IHC. Right bottom : Kaplan-Meier survival plot. Data analyzed by the log-rank test. F. Left : flow cytometry on tumor-infiltrating lymphocytes (TILs) at the conclusion of the experiment. 5 tumors/group. Right . All data is mean±SEM. Significance calculated using the unpaired t-test or a one-way ANOVA. * p

    Techniques Used: Inhibition, Mouse Assay, Expressing, Immunohistochemistry, Western Blot, Flow Cytometry, Cytometry

    2) Product Images from "Nitrosative Stress Induces Peroxiredoxin 1 Ubiquitination During Ischemic Insult via E6AP Activation in Endothelial Cells Both In Vitro and In Vivo"

    Article Title: Nitrosative Stress Induces Peroxiredoxin 1 Ubiquitination During Ischemic Insult via E6AP Activation in Endothelial Cells Both In Vitro and In Vivo

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2013.5381

    The ubiquitination of Prx1 in OGD-treated HBMEC. (A) The time-dependent change of OGD-induced Prx1 and ubiquitin expression in HBMECs was detected by Western blot. (B) Densitometry of the Western blots for (A) was normalized by the level of β-actin
    Figure Legend Snippet: The ubiquitination of Prx1 in OGD-treated HBMEC. (A) The time-dependent change of OGD-induced Prx1 and ubiquitin expression in HBMECs was detected by Western blot. (B) Densitometry of the Western blots for (A) was normalized by the level of β-actin

    Techniques Used: Expressing, Western Blot

    3) Product Images from "Tissue inhibitor of metalloproteinase 2 inhibits activation of the β-catenin signaling in melanoma cells"

    Article Title: Tissue inhibitor of metalloproteinase 2 inhibits activation of the β-catenin signaling in melanoma cells

    Journal: Cell Cycle

    doi: 10.1080/15384101.2015.1030557

    Distribution of β-catenin and cell growth and proliferation in A2058, T2R-7, and T2-1 cell lines. ( A ) Distribution of β-catenin by immunostaining. Blue: DAPI; Red: β-catenin. Green arrow shows small amount of β-catenin at nuclear area. ( B ) Cell growth curve. ( C ) Cell proliferation measured by the MTT. Data are expressed as mean ± SD. *P
    Figure Legend Snippet: Distribution of β-catenin and cell growth and proliferation in A2058, T2R-7, and T2-1 cell lines. ( A ) Distribution of β-catenin by immunostaining. Blue: DAPI; Red: β-catenin. Green arrow shows small amount of β-catenin at nuclear area. ( B ) Cell growth curve. ( C ) Cell proliferation measured by the MTT. Data are expressed as mean ± SD. *P

    Techniques Used: Immunostaining, MTT Assay

    TIMP-2 inhibits β-catenin induced by lithium chloride. ( A ) Immunoblot of β-catenin and p-c-Myc (Ser62) in A2058 (A), A2058T2R-7 (R), and A2058T2-1 (T) cell lines treated with 20 mM lithium chloride (LiCl) 30h. Densitometry of β-catenin and p-c-Myc in cells treated with LiCl. ( B ) Western blot analysis of β-catenin levels in cytosolic (Cy) and nuclear (Nu) extracts isolated from cells. SP1 serves as a nuclear protein loading control. Densitometry of nuclear β-catenin. Data are reported as mean ± SD of 3 independent experiments. ( C ) Distribution of β-catenin by immunostaining in A, R and T cells treated with 20mM LiCl for 30h. Blue: DAPI; Red: β-catenin. ( D ) TIMP-2 inhibited Wnt/T cell factor (TCF) responsive reporter in A, R, T cell lines treated with 20mMLiCl 30h.Luciferase activity was normalized to the internal control, set A2058 as 1. ( E ) Cell Proliferation measured by the MTT.
    Figure Legend Snippet: TIMP-2 inhibits β-catenin induced by lithium chloride. ( A ) Immunoblot of β-catenin and p-c-Myc (Ser62) in A2058 (A), A2058T2R-7 (R), and A2058T2-1 (T) cell lines treated with 20 mM lithium chloride (LiCl) 30h. Densitometry of β-catenin and p-c-Myc in cells treated with LiCl. ( B ) Western blot analysis of β-catenin levels in cytosolic (Cy) and nuclear (Nu) extracts isolated from cells. SP1 serves as a nuclear protein loading control. Densitometry of nuclear β-catenin. Data are reported as mean ± SD of 3 independent experiments. ( C ) Distribution of β-catenin by immunostaining in A, R and T cells treated with 20mM LiCl for 30h. Blue: DAPI; Red: β-catenin. ( D ) TIMP-2 inhibited Wnt/T cell factor (TCF) responsive reporter in A, R, T cell lines treated with 20mMLiCl 30h.Luciferase activity was normalized to the internal control, set A2058 as 1. ( E ) Cell Proliferation measured by the MTT.

    Techniques Used: Western Blot, Isolation, Immunostaining, Luciferase, Activity Assay, MTT Assay

    TIMP-2 affects the total, phosphorylated, and ubiquitinated β-catenin in A2058 cells. ( A ) β-catenin protein expression in human melanoma cell lines: parental A2058 expressing, A2058T2R-7 (T2R-7) under-expressing and A2058T2-1 (T2-1) over-expressing TIMP-2.Densitometry of β-catenin. ( B ) Immunoblot of phospho-β-catenin in A2058, T2R-7 and T2-1 cells. Densitometry of p-β-catenin (ser 552). ( C ) Immunoblot of total and phosphor-AKT in A2058, T2R-7 and T2-1 cells.Data are expressed as mean ± SD. *P
    Figure Legend Snippet: TIMP-2 affects the total, phosphorylated, and ubiquitinated β-catenin in A2058 cells. ( A ) β-catenin protein expression in human melanoma cell lines: parental A2058 expressing, A2058T2R-7 (T2R-7) under-expressing and A2058T2-1 (T2-1) over-expressing TIMP-2.Densitometry of β-catenin. ( B ) Immunoblot of phospho-β-catenin in A2058, T2R-7 and T2-1 cells. Densitometry of p-β-catenin (ser 552). ( C ) Immunoblot of total and phosphor-AKT in A2058, T2R-7 and T2-1 cells.Data are expressed as mean ± SD. *P

    Techniques Used: Expressing

    TIMP-2 and E-cadherin. ( A ) E-cadherin and N-cadherin protein expression in human melanoma cell lines. Densitometry of E-cadherin v.s. β-actin was measured. Data are expressed as mean ± SD. *P
    Figure Legend Snippet: TIMP-2 and E-cadherin. ( A ) E-cadherin and N-cadherin protein expression in human melanoma cell lines. Densitometry of E-cadherin v.s. β-actin was measured. Data are expressed as mean ± SD. *P

    Techniques Used: Expressing

    4) Product Images from "β1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells"

    Article Title: β1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003082

    AMPK activation enhances the interaction between β 1 Pix and 14-3-3 proteins but does not prevent binding of Nedd4-2 to 14-3-3 in mpkCCD c14 cells. A , representative immunoblot ( Input ) and co-IP results of β 1 Pix, Nedd4-2, and 14-3-3 proteins
    Figure Legend Snippet: AMPK activation enhances the interaction between β 1 Pix and 14-3-3 proteins but does not prevent binding of Nedd4-2 to 14-3-3 in mpkCCD c14 cells. A , representative immunoblot ( Input ) and co-IP results of β 1 Pix, Nedd4-2, and 14-3-3 proteins

    Techniques Used: Activation Assay, Binding Assay, Co-Immunoprecipitation Assay

    β 1 Pix knockdown blunts ENaC inhibition by AMPK. A and B , mCCD cl1 cells were stably transduced with either a scrambled control shRNA ( A ) or shRNA directed against β 1 Pix ( B ). EVOM studies were performed on polarized cells before and after
    Figure Legend Snippet: β 1 Pix knockdown blunts ENaC inhibition by AMPK. A and B , mCCD cl1 cells were stably transduced with either a scrambled control shRNA ( A ) or shRNA directed against β 1 Pix ( B ). EVOM studies were performed on polarized cells before and after

    Techniques Used: Inhibition, Stable Transfection, Transduction, shRNA

    Inducible V5-tagged WT β 1 Pix or β 1 Pix-Δ602–611 expression modulates ENaC activity in stably transfected mpkCCD c14 cells. A , EVOM studies were performed in the absence (−) or presence (+) of Dox induction (2 μg/ml)
    Figure Legend Snippet: Inducible V5-tagged WT β 1 Pix or β 1 Pix-Δ602–611 expression modulates ENaC activity in stably transfected mpkCCD c14 cells. A , EVOM studies were performed in the absence (−) or presence (+) of Dox induction (2 μg/ml)

    Techniques Used: Expressing, Activity Assay, Stable Transfection, Transfection

    β 1 Pix is necessary for the inhibition of ENaC by AMPK. A , overlays of typical macroscopic current traces before and after 10-μ m amiloride treatment from whole-cell patch-clamped CHO cells expressing mouse ENaC (mENaC) with or without WT
    Figure Legend Snippet: β 1 Pix is necessary for the inhibition of ENaC by AMPK. A , overlays of typical macroscopic current traces before and after 10-μ m amiloride treatment from whole-cell patch-clamped CHO cells expressing mouse ENaC (mENaC) with or without WT

    Techniques Used: Inhibition, Expressing

    Proposed model for the roles of Nedd4-2 and β 1 Pix in the regulation of ENaC by AMPK. Activation of AMPK promotes AMPK-mediated phosphorylation of Nedd4-2 at Ser-328 ( 1 ) and also promotes the binding of β 1 Pix to the Nedd4-2–14-3-3
    Figure Legend Snippet: Proposed model for the roles of Nedd4-2 and β 1 Pix in the regulation of ENaC by AMPK. Activation of AMPK promotes AMPK-mediated phosphorylation of Nedd4-2 at Ser-328 ( 1 ) and also promotes the binding of β 1 Pix to the Nedd4-2–14-3-3

    Techniques Used: Activation Assay, Binding Assay

    β 1 Pix regulates AMPK-dependent Nedd4-2–14-3-3 association and Nedd4-2 cellular stability in mCCD cl1 cells. A , representative immunoblot ( Input ) and co-IP results of β 1 Pix, Nedd4-2, and 14-3-3 proteins in total lysates from polarized
    Figure Legend Snippet: β 1 Pix regulates AMPK-dependent Nedd4-2–14-3-3 association and Nedd4-2 cellular stability in mCCD cl1 cells. A , representative immunoblot ( Input ) and co-IP results of β 1 Pix, Nedd4-2, and 14-3-3 proteins in total lysates from polarized

    Techniques Used: Co-Immunoprecipitation Assay

    5) Product Images from "Targeting NEDD8-activating enzyme is a new approach to treat canine diffuse large B-cell lymphoma"

    Article Title: Targeting NEDD8-activating enzyme is a new approach to treat canine diffuse large B-cell lymphoma

    Journal: Veterinary and comparative oncology

    doi: 10.1111/vco.12428

    Pevonedistat increases apoptosis in canine DLBCL. A, Representative gating strategy for flow cytometric analysis of pevonedistat-treated cells for early apoptosis (Annexin V + , SYTOX red − ), late apoptosis (annexin V + , SYTOX red + ) and healthy cells (Annexin V − , SYTOX red − ). B, Quantification of healthy, early apoptotic and late apoptotic CLBL-1. C, Western blot for cleaved caspase 3. CLBL-1 cells were treated with DMSO or pevonedistat (0.1–0.7 μM) for 6, 12 and 24 hours. Cell lysates were immunoblotted for cleaved caspase3 expression. β-actin was used as a loading control. D, Representative gating strategy for flow cytometric analysis of pevonedistat-treated primary canine DLBCL samples and quantification of early apoptotic cells. Early apoptotic cells were gated as Annexin V + DAPI − , late apoptotic cells were gated as Annexin V + DAPI + , and healthy cells were gated as Annexin V − DAPI − . ** P
    Figure Legend Snippet: Pevonedistat increases apoptosis in canine DLBCL. A, Representative gating strategy for flow cytometric analysis of pevonedistat-treated cells for early apoptosis (Annexin V + , SYTOX red − ), late apoptosis (annexin V + , SYTOX red + ) and healthy cells (Annexin V − , SYTOX red − ). B, Quantification of healthy, early apoptotic and late apoptotic CLBL-1. C, Western blot for cleaved caspase 3. CLBL-1 cells were treated with DMSO or pevonedistat (0.1–0.7 μM) for 6, 12 and 24 hours. Cell lysates were immunoblotted for cleaved caspase3 expression. β-actin was used as a loading control. D, Representative gating strategy for flow cytometric analysis of pevonedistat-treated primary canine DLBCL samples and quantification of early apoptotic cells. Early apoptotic cells were gated as Annexin V + DAPI − , late apoptotic cells were gated as Annexin V + DAPI + , and healthy cells were gated as Annexin V − DAPI − . ** P

    Techniques Used: Flow Cytometry, Western Blot, Expressing

    Pevonedistat treatment inhibits NF-κB pathway. A, CLBL-1 cells were treated with DMSO or pevonedistat (0.1–0.7 μM) for 1, 4, 12, and 24 hours. Cell lysates were immunoblotted for p-IκBα. β-actin was used as a loading control. B, CLBL-1 cells were treated with IC 90 concentration (0.58 μM) of pevonedistat for 1, 3, 12 and 24 hours, NF-κB pathway target gene expressions were measured by qRT-PCR and were compared with the gene expression levels in cells treated with DMSO. Gene expression level in DMSO-treated cells were set as ddCt = 0. C, Canine primary DLBCL cells were treated with pevonedistat for 24 hours, and NF-κB pathway target gene expressions were compared with DMSO control. Gene expression level in DMSO-treated cells were set as ddCt = 0. Each lane represents one patient sample
    Figure Legend Snippet: Pevonedistat treatment inhibits NF-κB pathway. A, CLBL-1 cells were treated with DMSO or pevonedistat (0.1–0.7 μM) for 1, 4, 12, and 24 hours. Cell lysates were immunoblotted for p-IκBα. β-actin was used as a loading control. B, CLBL-1 cells were treated with IC 90 concentration (0.58 μM) of pevonedistat for 1, 3, 12 and 24 hours, NF-κB pathway target gene expressions were measured by qRT-PCR and were compared with the gene expression levels in cells treated with DMSO. Gene expression level in DMSO-treated cells were set as ddCt = 0. C, Canine primary DLBCL cells were treated with pevonedistat for 24 hours, and NF-κB pathway target gene expressions were compared with DMSO control. Gene expression level in DMSO-treated cells were set as ddCt = 0. Each lane represents one patient sample

    Techniques Used: Concentration Assay, Quantitative RT-PCR, Expressing

    Pevonedistat treatment causes a G1-phase cell cycle arrest. A, Representative gating strategy for Ki67/DAPI cell proliferation assay. Cells in G0 phase were defined as Ki67 − DAPI − . Cells in G1 phase were defined as Ki67 + DAPI − . Cells in S/G2/M phase were defined as K67 + DAPI + . B, Quantification of CLBL-1 cells in G0, G1 and S/G2/M phases 72 hours post-treatment. C , CLBL-1 cells were treated with DMSO or pevonedistat (0.1–0.7 μM) for 6, 12 and 24 hours. Cell lysates were immunoblotted for p-H2AX. β-actin was used as a loading control. * P
    Figure Legend Snippet: Pevonedistat treatment causes a G1-phase cell cycle arrest. A, Representative gating strategy for Ki67/DAPI cell proliferation assay. Cells in G0 phase were defined as Ki67 − DAPI − . Cells in G1 phase were defined as Ki67 + DAPI − . Cells in S/G2/M phase were defined as K67 + DAPI + . B, Quantification of CLBL-1 cells in G0, G1 and S/G2/M phases 72 hours post-treatment. C , CLBL-1 cells were treated with DMSO or pevonedistat (0.1–0.7 μM) for 6, 12 and 24 hours. Cell lysates were immunoblotted for p-H2AX. β-actin was used as a loading control. * P

    Techniques Used: Proliferation Assay

    6) Product Images from "SIV-Induced Immune Activation and Metabolic Alterations in the Dorsal Root Ganglia During Acute Infection"

    Article Title: SIV-Induced Immune Activation and Metabolic Alterations in the Dorsal Root Ganglia During Acute Infection

    Journal: Journal of Neuropathology and Experimental Neurology

    doi: 10.1093/jnen/nly111

    Quantification of DRG proteins. Representative Western blot showing DRG homogenates from 2 uninfected and 2 7d SIV-infected macaques run in duplicate. Protein expression levels were measured by densitometry and normalized to in-lane β-actin.
    Figure Legend Snippet: Quantification of DRG proteins. Representative Western blot showing DRG homogenates from 2 uninfected and 2 7d SIV-infected macaques run in duplicate. Protein expression levels were measured by densitometry and normalized to in-lane β-actin.

    Techniques Used: Western Blot, Infection, Expressing

    7) Product Images from "Synthesis and Antileukemic Activities of Piperlongumine and HDAC Inhibitor Hybrids against Acute Myeloid Leukemia Cells"

    Article Title: Synthesis and Antileukemic Activities of Piperlongumine and HDAC Inhibitor Hybrids against Acute Myeloid Leukemia Cells

    Journal: Journal of medicinal chemistry

    doi: 10.1021/acs.jmedchem.6b00772

    PL-HDACis 1 – 58 , 3 – 35 , 3 – 31 , and 3 – 98 demonstrate HDACi activity. U937 cells were treated with 1 μ M of SAHA, PL, PL +SAHA, 1 – 58 , 3 – 35 , 3 – 31 , 3 – 98 , 35 , 36 , 27 , or 28 for 4 h. Whole cell lysates were subjected to Western blotting. Western blots were probed with anti–Ac-H4 (acetyl-histone H4), -H4, -Ac-Tubulin, or β -actin antibody.
    Figure Legend Snippet: PL-HDACis 1 – 58 , 3 – 35 , 3 – 31 , and 3 – 98 demonstrate HDACi activity. U937 cells were treated with 1 μ M of SAHA, PL, PL +SAHA, 1 – 58 , 3 – 35 , 3 – 31 , 3 – 98 , 35 , 36 , 27 , or 28 for 4 h. Whole cell lysates were subjected to Western blotting. Western blots were probed with anti–Ac-H4 (acetyl-histone H4), -H4, -Ac-Tubulin, or β -actin antibody.

    Techniques Used: Activity Assay, Western Blot

    8) Product Images from "The Adiponectin Receptor Agonist AdipoRon Ameliorates Diabetic Nephropathy in a Model of Type 2 Diabetes"

    Article Title: The Adiponectin Receptor Agonist AdipoRon Ameliorates Diabetic Nephropathy in a Model of Type 2 Diabetes

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2017060627

    AdipoRon activates the CaMKK β /phosphorylated Ser 431 LKB1/phosphorylated Thr 172 AMPK/PPAR α pathway by increasing intrarenal AdipoR1/AdipoR2 expression in db/db mice. (A–C) Representative images of immunofluorescence staining and quantitative
    Figure Legend Snippet: AdipoRon activates the CaMKK β /phosphorylated Ser 431 LKB1/phosphorylated Thr 172 AMPK/PPAR α pathway by increasing intrarenal AdipoR1/AdipoR2 expression in db/db mice. (A–C) Representative images of immunofluorescence staining and quantitative

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining

    9) Product Images from "Identification of Modulators That Activate the Constitutive Androstane Receptor From the Tox21 10K Compound Library"

    Article Title: Identification of Modulators That Activate the Constitutive Androstane Receptor From the Tox21 10K Compound Library

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfy242

    Protein induction in HPH of CYP2B6 and CYP3A4. In separate experiments, induction of CYP2B6 and CYP3A4 proteins were detected in an immunoblotting assay for HPH133 (A) and HPH139 (B). Membranes were soaked in primary antibodies at the following concentrations: 1:1000, 1:5000, and 1:30 000 for CYP2B6, CYP3A4, and β-actin, respectively. The secondary antibody was used at a concentration of 1:4000. NIT, nitazoxanide; AXI, axitinib; 2-AM, 2-aminoanthraquinone; NET, neticonazole; FRE, frentizole; DIP, diphenamid; PHE, phenothrin; RIM, rimcazole.
    Figure Legend Snippet: Protein induction in HPH of CYP2B6 and CYP3A4. In separate experiments, induction of CYP2B6 and CYP3A4 proteins were detected in an immunoblotting assay for HPH133 (A) and HPH139 (B). Membranes were soaked in primary antibodies at the following concentrations: 1:1000, 1:5000, and 1:30 000 for CYP2B6, CYP3A4, and β-actin, respectively. The secondary antibody was used at a concentration of 1:4000. NIT, nitazoxanide; AXI, axitinib; 2-AM, 2-aminoanthraquinone; NET, neticonazole; FRE, frentizole; DIP, diphenamid; PHE, phenothrin; RIM, rimcazole.

    Techniques Used: Concentration Assay

    10) Product Images from "Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma"

    Article Title: Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma

    Journal: Leukemia

    doi: 10.1038/s41375-019-0467-z

    WL40 blocks proteasome-mediated protein degradation without inhibiting proteasome proteolytic activities. a MM.1S cells were treated with DMSO control, bortezomib, or WL40 at indicated concentration for 3 h; protein lysates were analyzed for proteasome activities (CT-L, chymotrypsin-like; T-L, trypsin-like; C-L, caspase-like). The percentage of proteasome activity was normalized to DMSO control (mean ± SD; n = 3). b Recombinant 20S proteasome was incubated with DMSO, bortezomib, or WL40 for 30 min, followed by assessment of proteasome activities. Bar graph shows percent pro-teasome activity after normalization with DMSO control (mean ± SD; n = 3). c MM.1S cells were pretreated with MG132 (10 µM) for 1 h, followed by addition of WL40 (400 nM) for 8 h. As a positive control for WL40-induced Rpn13 degradation, cells were also treated with WL40 alone for 8 h. Total protein lysates were subjected to immunoblots for Rpn13 and α-tubulin. d RPMI-8226 cells were treated with WL40 (400 nM) for 16 h; protein lysates were subjected to immuno-blot analysis using anti-Rpn13 or anti-β-actin Abs. e ANBL6.BR cells were treated with WL40 (400 nM) or RA190 (500 nM) for 16 h; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-β-actin Abs. f (Left panel) MM.1S cells were treated with DMSO control, bortezomib (5 nM), RA190 (300 nM), or WL40 (200 nM) for indicated time periods; protein lysates were subjected to immunoblot analysis using antipolyubiquitin or anti-β-actin Abs. f (Right panel) ANBL6.BR cells were treated with DMSO control or WL40 (1 µM) for 6 h; protein lysates were subjected to immunoblot analysis using antipolyubiquitin or anti-β-actin Abs
    Figure Legend Snippet: WL40 blocks proteasome-mediated protein degradation without inhibiting proteasome proteolytic activities. a MM.1S cells were treated with DMSO control, bortezomib, or WL40 at indicated concentration for 3 h; protein lysates were analyzed for proteasome activities (CT-L, chymotrypsin-like; T-L, trypsin-like; C-L, caspase-like). The percentage of proteasome activity was normalized to DMSO control (mean ± SD; n = 3). b Recombinant 20S proteasome was incubated with DMSO, bortezomib, or WL40 for 30 min, followed by assessment of proteasome activities. Bar graph shows percent pro-teasome activity after normalization with DMSO control (mean ± SD; n = 3). c MM.1S cells were pretreated with MG132 (10 µM) for 1 h, followed by addition of WL40 (400 nM) for 8 h. As a positive control for WL40-induced Rpn13 degradation, cells were also treated with WL40 alone for 8 h. Total protein lysates were subjected to immunoblots for Rpn13 and α-tubulin. d RPMI-8226 cells were treated with WL40 (400 nM) for 16 h; protein lysates were subjected to immuno-blot analysis using anti-Rpn13 or anti-β-actin Abs. e ANBL6.BR cells were treated with WL40 (400 nM) or RA190 (500 nM) for 16 h; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-β-actin Abs. f (Left panel) MM.1S cells were treated with DMSO control, bortezomib (5 nM), RA190 (300 nM), or WL40 (200 nM) for indicated time periods; protein lysates were subjected to immunoblot analysis using antipolyubiquitin or anti-β-actin Abs. f (Right panel) ANBL6.BR cells were treated with DMSO control or WL40 (1 µM) for 6 h; protein lysates were subjected to immunoblot analysis using antipolyubiquitin or anti-β-actin Abs

    Techniques Used: Concentration Assay, Activity Assay, Recombinant, Incubation, Positive Control, Western Blot

    Design and characterization of Rpn13 degrader WL40. a WL40 was created by linking the Rpn13 inhibitor RA190 to the IMiD thalidomide as a ligand for the CRBN E3 ligase via a PEG linker. b The cereblon AlphaScreen assay to measure the displacement of biotinylated-pomalidomide probe (triplicate means ± SD). c Schematic cartoon for the novel AlphaScreen assay to measure the binding activity of inhibitor with RPN13 proteins. d The RPN13 AlphaScreen assay to measure the binding activity of inhibitor with RPN13 proteins (triplicate means ± SD). e MM.1S cells were treated with WL40 or RA190 at the indicated concentrations and time periods; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-tubulin Abs. f MM.1S cells were treated with DMSO control, WL40 (400 nM), or RA190 (500 nM) for 16 h; cells were then washed and stained with Rpn13 Ab conjugated with Alexa Fluor 647, followed by flow cytometry analysis. Isotype Ab conjugated to Alexa Fluor 647 was used as control for nonspecific binding. Data were quantified using FACS Diva (BD Biosciences, USA) and FlowJo (FlowJo LLC, USA). g MM.1S-CRBN-KO cells were treated with WL40 (400 nM) or RA190 (500 nM) for 16 h; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-β-actin Abs. Inset: protein lysates from MM.1S. WT control and MM.1S-CRBN-KO cells were subjected to immunoblot analysis using anti-CRBN or anti-β-actin Abs. h Immunoblot showing the levels of Ub-GFP accumulation in a GFPu-1 reporter cell line treated with indicated concentrations of WL40 and RA190 for 16 h. Blots shown are representative of three independent experiments. i HCT116-WT and HCT116-CRISPR Rpn13-KO cells were treated with WL40 or RA190 at the indicated concentrations for 48 h, followed by assessment of cell viability using the WST assay (mean ± SD; p
    Figure Legend Snippet: Design and characterization of Rpn13 degrader WL40. a WL40 was created by linking the Rpn13 inhibitor RA190 to the IMiD thalidomide as a ligand for the CRBN E3 ligase via a PEG linker. b The cereblon AlphaScreen assay to measure the displacement of biotinylated-pomalidomide probe (triplicate means ± SD). c Schematic cartoon for the novel AlphaScreen assay to measure the binding activity of inhibitor with RPN13 proteins. d The RPN13 AlphaScreen assay to measure the binding activity of inhibitor with RPN13 proteins (triplicate means ± SD). e MM.1S cells were treated with WL40 or RA190 at the indicated concentrations and time periods; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-tubulin Abs. f MM.1S cells were treated with DMSO control, WL40 (400 nM), or RA190 (500 nM) for 16 h; cells were then washed and stained with Rpn13 Ab conjugated with Alexa Fluor 647, followed by flow cytometry analysis. Isotype Ab conjugated to Alexa Fluor 647 was used as control for nonspecific binding. Data were quantified using FACS Diva (BD Biosciences, USA) and FlowJo (FlowJo LLC, USA). g MM.1S-CRBN-KO cells were treated with WL40 (400 nM) or RA190 (500 nM) for 16 h; protein lysates were subjected to immunoblot analysis using anti-Rpn13 or anti-β-actin Abs. Inset: protein lysates from MM.1S. WT control and MM.1S-CRBN-KO cells were subjected to immunoblot analysis using anti-CRBN or anti-β-actin Abs. h Immunoblot showing the levels of Ub-GFP accumulation in a GFPu-1 reporter cell line treated with indicated concentrations of WL40 and RA190 for 16 h. Blots shown are representative of three independent experiments. i HCT116-WT and HCT116-CRISPR Rpn13-KO cells were treated with WL40 or RA190 at the indicated concentrations for 48 h, followed by assessment of cell viability using the WST assay (mean ± SD; p

    Techniques Used: Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Activity Assay, Staining, Flow Cytometry, Cytometry, FACS, CRISPR, WST Assay

    WL40 inhibits xenografted human MM cell growth and prolongs host survival. a Mice bearing human MM.1S MM tumors were treated with either vehicle control, WL40 (14.7 μM/kg; i. p.), or RA190 (26.8 μM/kg; i.p.) twice weekly for 18 days. Tumor volume (mean tumor volume ± SD in mm 3 , 10 mice/ group) versus time is shown. b Kaplan–Meier plots shows survival of mice. c Lysates of tumors harvested from control-, WL40- and RA190-treated mice were subjected to immunoblot analysis using antipolyubiquitin, anticleaved-caspase-8, or anti-β-actin Abs. d Tumor sections from vehicle control-, and WL40-treated mice were stained with antipolyubiquitin, Ki67, caspase-3 (cleaved form), and CD31 Abs. Scale bar, 10 μm
    Figure Legend Snippet: WL40 inhibits xenografted human MM cell growth and prolongs host survival. a Mice bearing human MM.1S MM tumors were treated with either vehicle control, WL40 (14.7 μM/kg; i. p.), or RA190 (26.8 μM/kg; i.p.) twice weekly for 18 days. Tumor volume (mean tumor volume ± SD in mm 3 , 10 mice/ group) versus time is shown. b Kaplan–Meier plots shows survival of mice. c Lysates of tumors harvested from control-, WL40- and RA190-treated mice were subjected to immunoblot analysis using antipolyubiquitin, anticleaved-caspase-8, or anti-β-actin Abs. d Tumor sections from vehicle control-, and WL40-treated mice were stained with antipolyubiquitin, Ki67, caspase-3 (cleaved form), and CD31 Abs. Scale bar, 10 μm

    Techniques Used: Mouse Assay, Staining

    11) Product Images from "Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress"

    Article Title: Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddy297

    Generation of iPSC-derived neuronal cultures from PSP patients. (A) Images of representative iPSCs expressing pluripotency markers, Oct3/4, Sox2 and Nanog. Scale bar is 100 μm. (B) EB cultures derived from iPSC were stained with endodermal marker, α-fetoprotein (AFP), mesodermal marker, smooth muscle actin and neuroectodermal markers, Map2b or β-III-tubulin. Scale bar is 50 μm. (C) Representative G-banded normal karyotype from an iPSC line. (D) Representative images of FACS-purified iPSC-derived neural stem cells expressing NSC marker Sox2. Scale bar is 50 μm. (E) Representative images of 4 week differentiated neural cultures immunofluorescently stained with neuronal markers Map2b, β-III-tubulin or astrocytic marker glial fibrillary acid protein. Scale bar is 50 or 25 μm as shown.
    Figure Legend Snippet: Generation of iPSC-derived neuronal cultures from PSP patients. (A) Images of representative iPSCs expressing pluripotency markers, Oct3/4, Sox2 and Nanog. Scale bar is 100 μm. (B) EB cultures derived from iPSC were stained with endodermal marker, α-fetoprotein (AFP), mesodermal marker, smooth muscle actin and neuroectodermal markers, Map2b or β-III-tubulin. Scale bar is 50 μm. (C) Representative G-banded normal karyotype from an iPSC line. (D) Representative images of FACS-purified iPSC-derived neural stem cells expressing NSC marker Sox2. Scale bar is 50 μm. (E) Representative images of 4 week differentiated neural cultures immunofluorescently stained with neuronal markers Map2b, β-III-tubulin or astrocytic marker glial fibrillary acid protein. Scale bar is 50 or 25 μm as shown.

    Techniques Used: Derivative Assay, Expressing, Staining, Marker, FACS, Purification

    12) Product Images from "Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription"

    Article Title: Human Cytomegalovirus DNA Polymerase Subunit UL44 Antagonizes Antiviral Immune Responses by Suppressing IRF3- and NF-κB-Mediated Transcription

    Journal: Journal of Virology

    doi: 10.1128/JVI.00181-19

    UL44 inhibits IRF3- and NF-κB-mediated transcription of antiviral genes. (A) Effects of UL44 on the activation of the IFN-β promoter, ISRE, and NF-κB mediated by various components. HEK293 cells (1 × 10 5 ) were transfected with the IFN-β promoter, ISRE, and the NF-κB reporter, UL44, and the indicated expression plasmids for 20 h before luciferase assays. (B) Effects of UL44 on HCMV-induced phosphorylation of downstream components. HFFs stably expressing UL44 (2 × 10 6 ) were either left untreated or infected with HCMV (MOI, 1) for the indicated times before immunoblot analysis. (C) Effects of UL44 on IFN-α- and IFN-β-induced phosphorylation of STAT1. HFFs stably expressing UL44 (2 × 10 6 ) were either left untreated or treated with IFN-α or IFN-β for the indicated times before immunoblot analysis. F, Flag. (D) Association of UL44 with IRF3, p65, and p50. HEK293T cells (5 × 10 6 ) were transfected with the indicated plasmids for 20 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. IP, immunoprecipitation. αF, anti-Flag. (E) The association of UL44 with IRF3, p65, and p50 is DNA independent. HEK293T cells (5 × 10 6 ) were transfected with the indicated plasmids for 20 h. Cells were either left untreated or treated with DNase I (2 μg/ml) for 2 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Association of endogenous UL44 with IRF3, p65, and p50 in HFFs. HFFs (1 × 10 7 ) either were left untreated or were infected with HCMV for the indicated times before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. Pre, preimmune sera. (G) UL44 binds to IRF3, IRF7, p65, and p50 in vitro . Purified GST and GST-UL44 were used to pull down transiently expressed Flag-TBK1, Flag-IRF3, Flag-IRF7, Flag-p65, and Flag-p50 as indicated.
    Figure Legend Snippet: UL44 inhibits IRF3- and NF-κB-mediated transcription of antiviral genes. (A) Effects of UL44 on the activation of the IFN-β promoter, ISRE, and NF-κB mediated by various components. HEK293 cells (1 × 10 5 ) were transfected with the IFN-β promoter, ISRE, and the NF-κB reporter, UL44, and the indicated expression plasmids for 20 h before luciferase assays. (B) Effects of UL44 on HCMV-induced phosphorylation of downstream components. HFFs stably expressing UL44 (2 × 10 6 ) were either left untreated or infected with HCMV (MOI, 1) for the indicated times before immunoblot analysis. (C) Effects of UL44 on IFN-α- and IFN-β-induced phosphorylation of STAT1. HFFs stably expressing UL44 (2 × 10 6 ) were either left untreated or treated with IFN-α or IFN-β for the indicated times before immunoblot analysis. F, Flag. (D) Association of UL44 with IRF3, p65, and p50. HEK293T cells (5 × 10 6 ) were transfected with the indicated plasmids for 20 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. IP, immunoprecipitation. αF, anti-Flag. (E) The association of UL44 with IRF3, p65, and p50 is DNA independent. HEK293T cells (5 × 10 6 ) were transfected with the indicated plasmids for 20 h. Cells were either left untreated or treated with DNase I (2 μg/ml) for 2 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Association of endogenous UL44 with IRF3, p65, and p50 in HFFs. HFFs (1 × 10 7 ) either were left untreated or were infected with HCMV for the indicated times before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. Pre, preimmune sera. (G) UL44 binds to IRF3, IRF7, p65, and p50 in vitro . Purified GST and GST-UL44 were used to pull down transiently expressed Flag-TBK1, Flag-IRF3, Flag-IRF7, Flag-p65, and Flag-p50 as indicated.

    Techniques Used: Activation Assay, Transfection, Expressing, Luciferase, Stable Transfection, Infection, Immunoprecipitation, In Vitro, Purification

    Inhibition of innate antiviral signaling by HCMV UL44. (A) UL44 inhibits cGAS-MITA-induced activation of the IFN-β promoter, ISRE, and NF-κB in a dose-dependent manner. HEK293 cells (1 × 10 5 ) were transfected with an IFN-β promoter (0.05 μg), ISRE (0.03 μg), or NF-κB (0.005 μg) luciferase reporter plasmid, as well as with expression plasmids for cGAS (0.01 μg) and MITA (0.01 μg) and increased amounts of the UL44 plasmid, for 20 h before luciferase assays. Rel. Luc. Act., relative luciferase activity. The lower blots show the expression levels of the transfected UL44 protein. (B) Effects of UL44 on IFN-γ-induced IRF1 promoter activation. HEK293 cells (1 × 10 5 ) were transfected with IRF1 promoter reporter (0.05 μg) and UL44 expression (0.05 μg) plasmids for 20 h. The cells were then either left untreated or treated with IFN-γ for 12 h before luciferase assays. Vec, vector; ns, not significant. The lower blots show the expression levels of the transfected UL44 plasmids. (C) Expression of UL44 in a stable HFF-UL44 cell line. Control HFFs (2 × 10 5 ) (HFF-Vec) or HFFs stably expressing UL44 (2 × 10 5 ) were collected. Immunoblot analyses were performed with the indicated antibodies. (D) UL44 inhibits HCMV-, HSV-1-, and SeV-induced transcription of antiviral genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were either left uninfected or infected with HCMV, HSV-1, or SeV (each at an MOI of 1) for 12 h before qPCR analysis. (E) Inhibition by UL44 of IFNB1 , ISG56 , TNF , and IL-6 transcription induced by UV-inactivated HCMV. Control cells or cells stably expressing UL44 (5 × 10 5 ) were infected with wild-type or UV-inactivated HCMV for the indicated times before qPCR analysis. (F) UL44 inhibits dsDNA- and dsRNA-induced transcription of antiviral genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were transfected with ISD (2 μg) or poly(I:C) (2 μg) for the indicated times before qPCR analysis. (G) Effects of U44 on TNF-α - induced transcription of the TNF , IL-6 , and CXCL2 genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were treated with TNF-α for the indicated times before qPCR analysis. (H) Effects of UL44 on IFN-γ-induced transcription of the GBP1 and IRF1 genes or IFN-β-induced transcription of the ISG56 and CXCL10 genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were either left untreated or treated with IFN-γ or IFN-β for the indicated times before immunoblot analyses were performed. Graphs show means ± SD ( n  = 3). Asterisks indicate significant differences (*, P
    Figure Legend Snippet: Inhibition of innate antiviral signaling by HCMV UL44. (A) UL44 inhibits cGAS-MITA-induced activation of the IFN-β promoter, ISRE, and NF-κB in a dose-dependent manner. HEK293 cells (1 × 10 5 ) were transfected with an IFN-β promoter (0.05 μg), ISRE (0.03 μg), or NF-κB (0.005 μg) luciferase reporter plasmid, as well as with expression plasmids for cGAS (0.01 μg) and MITA (0.01 μg) and increased amounts of the UL44 plasmid, for 20 h before luciferase assays. Rel. Luc. Act., relative luciferase activity. The lower blots show the expression levels of the transfected UL44 protein. (B) Effects of UL44 on IFN-γ-induced IRF1 promoter activation. HEK293 cells (1 × 10 5 ) were transfected with IRF1 promoter reporter (0.05 μg) and UL44 expression (0.05 μg) plasmids for 20 h. The cells were then either left untreated or treated with IFN-γ for 12 h before luciferase assays. Vec, vector; ns, not significant. The lower blots show the expression levels of the transfected UL44 plasmids. (C) Expression of UL44 in a stable HFF-UL44 cell line. Control HFFs (2 × 10 5 ) (HFF-Vec) or HFFs stably expressing UL44 (2 × 10 5 ) were collected. Immunoblot analyses were performed with the indicated antibodies. (D) UL44 inhibits HCMV-, HSV-1-, and SeV-induced transcription of antiviral genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were either left uninfected or infected with HCMV, HSV-1, or SeV (each at an MOI of 1) for 12 h before qPCR analysis. (E) Inhibition by UL44 of IFNB1 , ISG56 , TNF , and IL-6 transcription induced by UV-inactivated HCMV. Control cells or cells stably expressing UL44 (5 × 10 5 ) were infected with wild-type or UV-inactivated HCMV for the indicated times before qPCR analysis. (F) UL44 inhibits dsDNA- and dsRNA-induced transcription of antiviral genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were transfected with ISD (2 μg) or poly(I:C) (2 μg) for the indicated times before qPCR analysis. (G) Effects of U44 on TNF-α - induced transcription of the TNF , IL-6 , and CXCL2 genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were treated with TNF-α for the indicated times before qPCR analysis. (H) Effects of UL44 on IFN-γ-induced transcription of the GBP1 and IRF1 genes or IFN-β-induced transcription of the ISG56 and CXCL10 genes in HFFs. HFFs stably expressing UL44 (5 × 10 5 ) were either left untreated or treated with IFN-γ or IFN-β for the indicated times before immunoblot analyses were performed. Graphs show means ± SD ( n  = 3). Asterisks indicate significant differences (*, P

    Techniques Used: Inhibition, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Activated Clotting Time Assay, Activity Assay, Stable Transfection, Infection, Real-time Polymerase Chain Reaction

    13) Product Images from "Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA"

    Article Title: Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2017.0494

    Characterization of GFP mmRNA release from scaffold and in vitro transfection. (A) Cumulative mmRNA release from GFP mmRNA-loaded aligned nanofibrillar scaffolds expressed relative to the total recovered mmRNA ( n  = 3). Initial mmRNA loadings into scaffolds were 90 ng (1 × ); 270 ng (3 × ), or 450 ng (5 × ), ( n  = 3). ( B) Live fluorescence images of GFP one day after seeding of human fibroblasts on GFP mmRNA-releasing nanofibrillar scaffolds . (C) Immunoblot quantification of GFP expression in cell lysates after 1 day of transfection for varying initial loads of mmRNA. Graph shows GFP protein normalized to β-actin ( n  = 3). (D) Immunoblot quantification of GFP expression time course ( n  = 3). *Statistically significant relationship, compared to 1 × mmRNA loading at the same time point ( p
    Figure Legend Snippet: Characterization of GFP mmRNA release from scaffold and in vitro transfection. (A) Cumulative mmRNA release from GFP mmRNA-loaded aligned nanofibrillar scaffolds expressed relative to the total recovered mmRNA ( n  = 3). Initial mmRNA loadings into scaffolds were 90 ng (1 × ); 270 ng (3 × ), or 450 ng (5 × ), ( n  = 3). ( B) Live fluorescence images of GFP one day after seeding of human fibroblasts on GFP mmRNA-releasing nanofibrillar scaffolds . (C) Immunoblot quantification of GFP expression in cell lysates after 1 day of transfection for varying initial loads of mmRNA. Graph shows GFP protein normalized to β-actin ( n  = 3). (D) Immunoblot quantification of GFP expression time course ( n  = 3). *Statistically significant relationship, compared to 1 × mmRNA loading at the same time point ( p

    Techniques Used: In Vitro, Transfection, Fluorescence, Expressing

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    Produced:

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    Incubation:

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  • 88
    Millipore ãŽâ² actin
    TGFβ inhibition augments anti–CTLA-4 immunotherapy in an autochthonous BRAF V600E PTEN -/- melanoma model. A. Mice were treated with either anti–CTLA-4 (purple, 100 μg i.p.) or IgG isotype control (black, 100 μg i.p.) every 3 days when tumors reached 60–80 mm 3 . Left : tumor volumes monitored every 3 days. Right : representative tumor photos. 6 mice/group. Representative of 3 independent experiments. B. Left : resected Braf V600E Pten -/- melanoma tissues analyzed for TGFβ1 expression by IHC and whole tissue Western blots. IHC representative of 3 tumor specimens (40x). Right : Spearman correlation between tumor volume and <t>TGFβ1/β-actin</t> density ratios from Western blots. C. Phospho-SMAD2 (pSMAD2) Western blot analysis performed following TEW-7197 treatment (25 mg/kg daily p.o. x 2 weeks). tSMAD2: total SMAD2. Representative of 2 independent experiments. D. Left : mice were treated with TEW-7197 monotherapy (25 mg/kg p.o. daily). Tumor volumes were monitored every 3 days. 6 mice/group. Representative of 3 independent experiments. Center : final tumor weights. Right : representative photos of resected tumors. ns: non-significant. E. Left : mice were treated with either IgG isotype control/vehicle control, TEW-7197(25 mg/kg p.o. daily) monotherapy, or combination anti–CTLA-4 (100 μg i.p. every 3 days)/TEW-7197. Left : tumor volumes monitored every 3 days. 6 mice/group. Black arrow, treatment initiation. Representative of 2 independent experiments. Right top : lungs resected and enumerated for metastatic foci by staggered IHC. Right bottom : Kaplan-Meier survival plot. Data analyzed by the log-rank test. F. Left : flow cytometry on tumor-infiltrating lymphocytes (TILs) at the conclusion of the experiment. 5 tumors/group. Right . All data is mean±SEM. Significance calculated using the unpaired t-test or a one-way ANOVA. * p
    ãŽâ² Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore sema4d
    Live labeling with <t>sema4D</t> antibody shows change of outer retina sema4D in WT and RCS retina comparing after light onset. a Live sema4D antibody and secondary antibody labeling (shown in red) of whole mount WT retina 1 hour before and after light onset
    Sema4d, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore ãŽâ egfr mab
    ATP-mediated DUOX1 activation promotes <t>TGF-α</t> shedding in HBE1 cells. A, HBE1 cells were stimulated with either ATP or α-ASGM1 for 2 h, in the absence or presence of <t>α-EGFR</t> mAb (225; 4 μg/ml), and conditioned media were
    ãŽâ Egfr Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore mmp
    Dominant-negative ezrin-induced inhibition of migration and invasion: association with altered BCL-2/BAX rheostat, decreased <t>MMP</t> activity, and decreased α v β 3 integrin expression. A, The levels of BCL-2, BCL-X L , and BAX in LN-229 whole-cell lysates and of MMP-2, MMP-9, MT1-MMP, and <t>TIMP-2</t> in the supernatant were examined by immunoblot. MMP-2 activity in conditioned medium of neo, nter-ezrin-, and F353-ezrin-transfected LN-229 cells was examined by gelatin zymography. All blots are representative of experiments performed three times with similar results. B, α v β 3 and α 5 β 1 integrin expression were determined by flow cytometry. The curves for α V β 3 antibody ( bold line ) and control antibody ( dotted line ) are shown. As documented in the bottom panel , there was no change in α 5 β 1 integrin. In the bar graph, data are expressed as mean SFI values ( n = 3).
    Mmp, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TGFβ inhibition augments anti–CTLA-4 immunotherapy in an autochthonous BRAF V600E PTEN -/- melanoma model. A. Mice were treated with either anti–CTLA-4 (purple, 100 μg i.p.) or IgG isotype control (black, 100 μg i.p.) every 3 days when tumors reached 60–80 mm 3 . Left : tumor volumes monitored every 3 days. Right : representative tumor photos. 6 mice/group. Representative of 3 independent experiments. B. Left : resected Braf V600E Pten -/- melanoma tissues analyzed for TGFβ1 expression by IHC and whole tissue Western blots. IHC representative of 3 tumor specimens (40x). Right : Spearman correlation between tumor volume and TGFβ1/β-actin density ratios from Western blots. C. Phospho-SMAD2 (pSMAD2) Western blot analysis performed following TEW-7197 treatment (25 mg/kg daily p.o. x 2 weeks). tSMAD2: total SMAD2. Representative of 2 independent experiments. D. Left : mice were treated with TEW-7197 monotherapy (25 mg/kg p.o. daily). Tumor volumes were monitored every 3 days. 6 mice/group. Representative of 3 independent experiments. Center : final tumor weights. Right : representative photos of resected tumors. ns: non-significant. E. Left : mice were treated with either IgG isotype control/vehicle control, TEW-7197(25 mg/kg p.o. daily) monotherapy, or combination anti–CTLA-4 (100 μg i.p. every 3 days)/TEW-7197. Left : tumor volumes monitored every 3 days. 6 mice/group. Black arrow, treatment initiation. Representative of 2 independent experiments. Right top : lungs resected and enumerated for metastatic foci by staggered IHC. Right bottom : Kaplan-Meier survival plot. Data analyzed by the log-rank test. F. Left : flow cytometry on tumor-infiltrating lymphocytes (TILs) at the conclusion of the experiment. 5 tumors/group. Right . All data is mean±SEM. Significance calculated using the unpaired t-test or a one-way ANOVA. * p

    Journal: Cancer immunology research

    Article Title: Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma

    doi: 10.1158/2326-6066.CIR-18-0086

    Figure Lengend Snippet: TGFβ inhibition augments anti–CTLA-4 immunotherapy in an autochthonous BRAF V600E PTEN -/- melanoma model. A. Mice were treated with either anti–CTLA-4 (purple, 100 μg i.p.) or IgG isotype control (black, 100 μg i.p.) every 3 days when tumors reached 60–80 mm 3 . Left : tumor volumes monitored every 3 days. Right : representative tumor photos. 6 mice/group. Representative of 3 independent experiments. B. Left : resected Braf V600E Pten -/- melanoma tissues analyzed for TGFβ1 expression by IHC and whole tissue Western blots. IHC representative of 3 tumor specimens (40x). Right : Spearman correlation between tumor volume and TGFβ1/β-actin density ratios from Western blots. C. Phospho-SMAD2 (pSMAD2) Western blot analysis performed following TEW-7197 treatment (25 mg/kg daily p.o. x 2 weeks). tSMAD2: total SMAD2. Representative of 2 independent experiments. D. Left : mice were treated with TEW-7197 monotherapy (25 mg/kg p.o. daily). Tumor volumes were monitored every 3 days. 6 mice/group. Representative of 3 independent experiments. Center : final tumor weights. Right : representative photos of resected tumors. ns: non-significant. E. Left : mice were treated with either IgG isotype control/vehicle control, TEW-7197(25 mg/kg p.o. daily) monotherapy, or combination anti–CTLA-4 (100 μg i.p. every 3 days)/TEW-7197. Left : tumor volumes monitored every 3 days. 6 mice/group. Black arrow, treatment initiation. Representative of 2 independent experiments. Right top : lungs resected and enumerated for metastatic foci by staggered IHC. Right bottom : Kaplan-Meier survival plot. Data analyzed by the log-rank test. F. Left : flow cytometry on tumor-infiltrating lymphocytes (TILs) at the conclusion of the experiment. 5 tumors/group. Right . All data is mean±SEM. Significance calculated using the unpaired t-test or a one-way ANOVA. * p

    Article Snippet: Primary antibodies included TGFβ (Cell Signaling, cat. number 3711), total SMAD2 (Cell signaling, cat. number 5339), phospho-SMAD2 (S465/467; Cell signaling, cat. number 3108), β-actin (Millipore, cat. number MABT1333), and MMP-9 (Millipore, cat. number MABT171) and were used at 1:1000.

    Techniques: Inhibition, Mouse Assay, Expressing, Immunohistochemistry, Western Blot, Flow Cytometry, Cytometry

    Live labeling with sema4D antibody shows change of outer retina sema4D in WT and RCS retina comparing after light onset. a Live sema4D antibody and secondary antibody labeling (shown in red) of whole mount WT retina 1 hour before and after light onset

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: Live labeling with sema4D antibody shows change of outer retina sema4D in WT and RCS retina comparing after light onset. a Live sema4D antibody and secondary antibody labeling (shown in red) of whole mount WT retina 1 hour before and after light onset

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques: Labeling, Antibody Labeling

    Sema4D inhibits POS internalization without affecting POS binding. a Maximal projections of x-y confocal image stacks and a select x-z plane as indicated showing ZO-1 (shown in red), cell nuclei (shown in blue) and bound POS (shown in green) at the apical

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: Sema4D inhibits POS internalization without affecting POS binding. a Maximal projections of x-y confocal image stacks and a select x-z plane as indicated showing ZO-1 (shown in red), cell nuclei (shown in blue) and bound POS (shown in green) at the apical

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques: Binding Assay

    Sema4D inhibits POS internalization by preventing Rac1 activation during POS challenge . a Immunoblotting of phosphorylated AKT (60 kDa), AKT (60 kDa), RhoA (22 kDa), Rac1 (21 kDa) and porin (31 kDa) as loading control in hRPE after POS internalization

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: Sema4D inhibits POS internalization by preventing Rac1 activation during POS challenge . a Immunoblotting of phosphorylated AKT (60 kDa), AKT (60 kDa), RhoA (22 kDa), Rac1 (21 kDa) and porin (31 kDa) as loading control in hRPE after POS internalization

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques: Activation Assay

    Sema4D localizes to neural retina and the neighboring RPE in the rat eye while plexin B1 localizes to RPE only. Tissue samples were harvested 1 hour before light onset. a Immunoblotting detection of plxnB1 (200 kDa), sema4D (150 kDa), the RPE marker protein

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: Sema4D localizes to neural retina and the neighboring RPE in the rat eye while plexin B1 localizes to RPE only. Tissue samples were harvested 1 hour before light onset. a Immunoblotting detection of plxnB1 (200 kDa), sema4D (150 kDa), the RPE marker protein

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques: Marker

    Sema4D localizes to outer segments of cone photoreceptors . a Field shows isolated retina labeled live with sema4D antibody (shown in red) and fluorescent PNA marking cone outer segments (shown in green). Scale bar: 10 μm. b Field shows isolated

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: Sema4D localizes to outer segments of cone photoreceptors . a Field shows isolated retina labeled live with sema4D antibody (shown in red) and fluorescent PNA marking cone outer segments (shown in green). Scale bar: 10 μm. b Field shows isolated

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques: Isolation, Labeling

    Sema4D KO RPE harbors excess POS phagosomes at the diurnal burst of phagocytosis. a Images show comparable retinal histology in sema4D KO and WT eyes with all retinal layers present: Ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: Sema4D KO RPE harbors excess POS phagosomes at the diurnal burst of phagocytosis. a Images show comparable retinal histology in sema4D KO and WT eyes with all retinal layers present: Ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques:

    PlxnB1 activity and sema4D protein levels decrease in WT but not RCS rat eyes at the peak time of RPE phagocytosis 1 hour after light onset. a Immunoblot of plxnB1 (200 kDa), sema4D (150 kDa) and porin (31 kDa) of WT eye samples dissected 1 hour before

    Journal: Molecular neurobiology

    Article Title: Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

    doi: 10.1007/s12035-017-0649-5

    Figure Lengend Snippet: PlxnB1 activity and sema4D protein levels decrease in WT but not RCS rat eyes at the peak time of RPE phagocytosis 1 hour after light onset. a Immunoblot of plxnB1 (200 kDa), sema4D (150 kDa) and porin (31 kDa) of WT eye samples dissected 1 hour before

    Article Snippet: Freshly dissected whole neural retinas were then incubated in PBS-CM with non-immune IgG or antibody to sema4D (15 μg/ml) for 1 h on ice followed by 3 washes with PBS-CM and incubation with AlexaFluor-conjugated secondary antibodies, peanut agglutinin or wheat germ agglutinin lectins.

    Techniques: Activity Assay

    ATP-mediated DUOX1 activation promotes TGF-α shedding in HBE1 cells. A, HBE1 cells were stimulated with either ATP or α-ASGM1 for 2 h, in the absence or presence of α-EGFR mAb (225; 4 μg/ml), and conditioned media were

    Journal: The Journal of Biological Chemistry

    Article Title: ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli *

    doi: 10.1074/jbc.M809761200

    Figure Lengend Snippet: ATP-mediated DUOX1 activation promotes TGF-α shedding in HBE1 cells. A, HBE1 cells were stimulated with either ATP or α-ASGM1 for 2 h, in the absence or presence of α-EGFR mAb (225; 4 μg/ml), and conditioned media were

    Article Snippet: To avoid TGF-α binding to EGFR, cells were in some cases pretreated with an α-EGFR mAb (225; 4 μg/ml; Calbiochem) for 30 min prior to stimulation.

    Techniques: Activation Assay

    Schematic representation of DUOX1 involvement in TLR-mediated IL-8 production in response to bacterial stimuli. Cell stimulation with LPS or α-ASGM1 mediates release of ATP, which activates EGFR-ERK1/2-NF-κB signaling cascades which is

    Journal: The Journal of Biological Chemistry

    Article Title: ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli *

    doi: 10.1074/jbc.M809761200

    Figure Lengend Snippet: Schematic representation of DUOX1 involvement in TLR-mediated IL-8 production in response to bacterial stimuli. Cell stimulation with LPS or α-ASGM1 mediates release of ATP, which activates EGFR-ERK1/2-NF-κB signaling cascades which is

    Article Snippet: To avoid TGF-α binding to EGFR, cells were in some cases pretreated with an α-EGFR mAb (225; 4 μg/ml; Calbiochem) for 30 min prior to stimulation.

    Techniques: Cell Stimulation

    Dominant-negative ezrin-induced inhibition of migration and invasion: association with altered BCL-2/BAX rheostat, decreased MMP activity, and decreased α v β 3 integrin expression. A, The levels of BCL-2, BCL-X L , and BAX in LN-229 whole-cell lysates and of MMP-2, MMP-9, MT1-MMP, and TIMP-2 in the supernatant were examined by immunoblot. MMP-2 activity in conditioned medium of neo, nter-ezrin-, and F353-ezrin-transfected LN-229 cells was examined by gelatin zymography. All blots are representative of experiments performed three times with similar results. B, α v β 3 and α 5 β 1 integrin expression were determined by flow cytometry. The curves for α V β 3 antibody ( bold line ) and control antibody ( dotted line ) are shown. As documented in the bottom panel , there was no change in α 5 β 1 integrin. In the bar graph, data are expressed as mean SFI values ( n = 3).

    Journal: The Journal of Neuroscience

    Article Title: Ezrin-Dependent Promotion of Glioma Cell Clonogenicity, Motility, and Invasion Mediated by BCL-2 and Transforming Growth Factor-β2

    doi: 10.1523/JNEUROSCI.21-10-03360.2001

    Figure Lengend Snippet: Dominant-negative ezrin-induced inhibition of migration and invasion: association with altered BCL-2/BAX rheostat, decreased MMP activity, and decreased α v β 3 integrin expression. A, The levels of BCL-2, BCL-X L , and BAX in LN-229 whole-cell lysates and of MMP-2, MMP-9, MT1-MMP, and TIMP-2 in the supernatant were examined by immunoblot. MMP-2 activity in conditioned medium of neo, nter-ezrin-, and F353-ezrin-transfected LN-229 cells was examined by gelatin zymography. All blots are representative of experiments performed three times with similar results. B, α v β 3 and α 5 β 1 integrin expression were determined by flow cytometry. The curves for α V β 3 antibody ( bold line ) and control antibody ( dotted line ) are shown. As documented in the bottom panel , there was no change in α 5 β 1 integrin. In the bar graph, data are expressed as mean SFI values ( n = 3).

    Article Snippet: The following antibodies were used at the indicated concentrations: MMP-2 (72 kDa)/MMP-9 (92 kDa), tissue inhibitor of metalloproteinases-2 (TIMP-2) (21 kDa), membrane type-1 of MMP (MT-1-MMP) (66 kDa) (2 μg/ml; Oncogene, Calbiochem, Schwalbach, Germany), BCL-2 (26 kDa), BAX (21 kDa), and TGF-β1/2 (55 kDa) (2 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), anti-vesicular stomatitis virus-glycoprotein (VSVG) antibody, anti-phosphoserine antibody, and anti-phosphotyrosine antibody (1 μg/ml; Sigma), anti-Akt antibody (46 kDa), anti-focal adhesion kinase (FAK) antibody (125 kDa) (2 μg/ml; Transduction Laboratories, Lexington, KY), and anti-CD44 antibody (clone 2C5) (R & D Systems, Abingdon, UK).

    Techniques: Dominant Negative Mutation, Inhibition, Migration, Activity Assay, Expressing, Transfection, Zymography, Flow Cytometry, Cytometry